Pathogenicity of Beauveria bassiana and Metarhizium anisopliae to Anastrepha fraterculus (Diptera: Tephritidae) and Effects on Adult Longevity

Anastrepha fraterculus (Diptera: Tephritidae) is among the most important fruit pests in South America, and the use of entomopathogenic fungi is considered a promising alternative for its control. The objective of this work was to evaluate the pathogenicity of Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin on larvae and pupae of A. fraterculus, along with fungal effects on adult fly longevity. Fungal inoculations, fly larvae or pupae were placed in Petri dishes with 1 mL/plate, and the concentrations of 10, 15, 20 and 25 grams of commercial product/liter of water. Controls received water only. To evaluate the residual effect on adult flies, emerging adults were transferred to clean arenas and the adult longevity was monitored. Beauveria bassiana and M. anisopliae caused 93.3 and 96.7% larval mortality and 14.0 and 15.0% pupal mortality, respectively. The estimated LC50 and LC90 values were 22.56 and 40.87 g/L for B. bassiana, and of 23.45 and 42.02 g/L for M. anisopliae. Infected adult insects had shorter longevity than non-infected insects, with mean survival of 8.0 and 83.5 days for B. bassiana and M. anisopliae, respectively.


Introduction
The fruit fly Anastrepha fraterculus (Wiedemann 1830) (Diptera: Tephritidae) is among the most important fruit fly pests in South America, infesting more than 100 species of both native and exotic plants (Hendrichs, Vera, De Meyer, & Clarke, 2015;Zucchi, 2017).The fly damage can both direct, by the female during oviposition and larval development in the fruit, and indirect, due to penetration of secondary pathogens through oviposition injuries (Zart, Fernandes, & Botton, 2010).Among the fruit fly control techniques, the use of chemical insecticides is the most frequent, however, due to high toxicity these products are less desirable, long no-entry periods and low selectivity toward natural enemies (Garcia, Brida, Martins, Abeijon, & Lutinski, 2017).
An alternative control for these pests is the use of biological organisms which are efficient, have low environmental impacts and can be combined with other control techniques in an Integrated Pest Management Program (Lenteren, Bolckmans, Kohl, Ravensberg, & Urbaneja, 2017).Entomopathogenic fungi stand out as a control alternative due to easy application, efficient pest control in the short time, and safety toward man and the environment (Sinha, Choudhary, & Kumari, 2016).Among the most commonly used fungi in pest control are Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin (Ascomycota: Hypocreales) which can affect the different stages of the pest development (Butt, Coates, Dubovskiy, & Ratcliffe, 2016).
When in contact with the host, the fungal spores adhere to the surface of the cuticle, germinate and produce specialized structures (appressoria) that allow penetration of the integument through a combination of enzymes and mechanical forces (Ortiz-Urquiza & Keyhani, 2013).After penetration, fungal hyphae invade and proliferate in the host hemolymph, leading to insect mortality (Mora, Castilho, & Fraga, 2016).
In view of the damage that A. fraterculus causes to many economically important fruit species and the potential efficiency of entomopathogenic fungi as part of an Integrated Pest Manegement (IPM) approach, the present study aimed to evaluate the pathogenicity of B. bassiana and M. anisopliae on larvae and pupae of A. fraterculus and lasting effect on adult longevity.

Method
Insect populations: The experiments were carried out at the Laboratório de Ecologia de Insetos of the Federal University of Pelotas-UFPel, in the district of Capão do Leão, Rio Grande do Sul, Brazil.The larvae and pupae of A. fraterculus were obtained from the creation of insects from the Laboratory of Insect Biology-UFPel, kept at 25±1 ºC, and 70±10% relative humidity with 12 h photophase.The insects were transferred to Petri dishes (10 cm in diameter x 1.5 cm in height) containing moist cotton and artificial diet based on refined sugar, wheat germ and beer yeast (3:1:1) (Bionis® BIONIS ® YE MF and NS), according to the methodology Salles (1992) modified by Nunes et al. (2013).
Fungal isolates: The commercial B. bassiana (BOVERIL® WP PL63) and M. anisopliae (METARRIL® WP E9) wettable powder products were obtained from Koopert Biological Systems.Conidial viability tests were carried out at the Laboratório de Micologia of the Federal University of Pelotas-UFPel, using the microculture technique, adapted from França, Marques, Torres, and Oliveira (2006).The fungal products were diluted to the concentration of 1 × 10 4 conidia/mL of distilled water in concentration and 100 μL of the suspensions were inoculated on potato agar dextrose medium (PDA) in 9-cm Petri dishes.The Petri dishes were incubated at 25±1 °C, with 70±10% RH, in the dark until colony forming units (CFUs) were sufficiently grown to be counted.This procedure was repeated daily for four days for each isolate used.
Pathogenicity and lethal concentration (LC 50 and LC 90 ): Experiments were conducted in a complete randomized design with eight treatments and a control, all applied in 10 replicates, using fungal isolates following methodology by F. Q. Oliveira, Batista, Malaquias, Almeida, and R. Oliveira (2010).The commercial products were weighed in aliquots representing the concentrations recommended by the manufacturer for field application (10, 15, 20 and 25 grams of product/liter of water or 5.0 × 10 6 , 7.5 × 10 6 , 10.0 × 10 6 and 12.5 × 10 6 conidia/mL respectively).Products were transferred to flasks with 1 L of sterile water and agitated in vortex agitators until complete dilution.From the initial solution, aliquots of 17 μL were removed (recommended by the manufacturer) and were diluted in 1 mL of water (distilled and sterile) and inoculated into Petri dishes (9 cm) coated with two sterilized sheets of filter paper disks (autoclave at 1 atm and 121 °C for 30 minutes).The control consisted of 1 mL sterile distilled water per container applied in similar manner as the fungal treatments.After inoculation, 10 A. fraterculus larvae and/or pupae were inserted separately in each Petri dishes.Larvae (13-day old), corresponding to the stage in which the larva leaves the fruit towards the soil, and pupae with fully formed integument (1 day after pupation) were used in the experiments.The Petri dishes were sealed with PVC film and stored in incubator (25±1 °C, 70±10% RH, and complete darkness).Third instar larva and pupa mortality were evaluated daily until the complete emergence of the control adults (12 to 15 days).
In order to confirm mortality due to fungal infection, dead insects and non-viable pupae were removed from the inoculation Petri dish and were sterilized with sodium hypochlorite (1.0%), then alcohol (70.0%) followed by wash in sterile distilled water (Quesada-Moraga, Martin-Carballo, Garrido-Jurado, & Santiago-Alvarez, 2008).After asepsis, the insects were incubated in Petri dishes (9 cm) lined with two sheets of moist sterile filter paper (1 ml sterile distilled water).The Petri dishes with the insects were capped and sealed with plastic film and stored in incubator set at 25±1 °C, RH of 70±10%, and lights off to allow fungal development on the insect cadaver.

%) of
Pathogenicity test: All conidial concentrations of both isolates showed pathogenicity to A. fraterculus, with some differences seen as a function of the insect stage of development.In the highest concentration for inoculations to the larval stage, B. bassiana and M. anisopliae caused total mortality of up to 93.3% (±14.05) and 96.7% (±14.05) of insects, respectively (Table 1).Inoculations at the larval stage resulted in low larval mortality, with 10.0% (±16.09) for B. bassiana and 6.7% (±10.50) for M. anisopliae, which did not differ from the control (P = 0.600) (Table 1).Despite being infected, the insects advanced to later stages, in which the most mortality occurred.In pupae, B. bassiana caused 56.7% (±27.40) and M. anisopliae caused 53.3% (±23.30)mortality, and both treatments differed from the control (Table 1).In adults, the mortality was 36.7% (±18.90) with B. bassiana and 43.0%(±22.50) with M. anisopliae, both significantly different from the control (Table 1).
In inoculations carried out at the pupae stage, the higher mortality was 14.0% (±6.99) with B. bassiana, and 15.0% (±6.99) for M. anisopliae.Pupal inoculations did not cause infections to the insects at later stages of development (adult) (Table 2).
Lethal concentrations (LC 50 and LC 90 ): The LC 50 and LC 90 values for B. bassiana and M. anisiopliae did not differ significantly between the isolates, but there were significant differences between the developmental stages of the insect.After larval inoculations, the insects progressed to the pupal stage, and this stage was affected, with lower LC 50 and LC 90 (22.56 g/L and 40.87 g/L for B. bassiana, and 23.45 gr/L and 42.02 gr/L for M. anisopliae, respectively (Table 3).For inoculations at the pupal stage, higher product concentrations were required, with LC 50 's at 95.73 g/L and 83.13 g/L for B. bassiana and LC 90 's at 175.15 g/L and 150.90 g/L for M. anisopliae (Table 4).

Table 1 .
Confirmed mortality [mean ± standard deviation (SD)] in different developmental stages of Anastrepha fraterculus after treatment with fungal isolates Beauveria bassiana (Balsamo) and Metarhizium anisopliae (Metschnikoff) in the larval stage

Table 2 .
Confirmed mortality [mean ± standard deviation (SD)] in different developmental stages of Anastrepha fraterculus exposed to treatments with fungal isolates Beauveria bassiana (Balsamo) and Metarhizium anisopliae (Metschnikoff) in the pupal stage

Table 3 .
Lethal concentrations (LC 50 and LC 90 ) (grams/liter) (confidence intervals) of commercial fungal isolates of Beauveria bassiana and Metarhizium anisopliae to larvae, pupae and adults of Anastrepha fraterculus after treatments in the larval stage of the insect Note.The means followed by different letters within the same column are significantly different (P < 0.05). jas.ccsenet.